前列腺癌中N—myc下游调节基因-1启动子区甲基化状态的初步研究

Preliminary study of NDRG1 gene promoter methylation status in prostate cancer

目的检测前列腺癌中N-myc下游调节基因-1（N—mycdownstreamregu1atedgene-1，NDRG1）启动子区的甲基化状态，探讨甲基化转移酶抑制剂5-氮杂胞苷对NDRG1基因在前列腺癌细胞mRNA的表达及对细胞增殖的影响。方法2013年1月至2014年4月采用亚硫酸盐测序PCR法分别检测前列腺癌组织、良性前列腺增生（BPH）组织、前列腺癌细胞系（PC3、22RV1、LNCaP、DU145）和人正常前列腺细胞系RWPE-1中的NDRG1基因启动子区甲基化状态。用10~mo1／L5-氮杂胞苷分别作用于LNCaP和DU145细胞72h后，噻唑盐法分析5-氮杂胞苷对LNCaP和DU145细胞增殖的影响；RT．PCR法检测两种细胞系中NDRG1mRNA的表达。结果NDRG1基因在前列腺癌细胞系PC．3、22RV1、LNCaP和DU145中甲基化率分别为（24．8±3．3）％、（36．2±2．5）％、（48．6±2．8）％、（69．5±1．7）％，人正常前列腺细胞系RWPE-1甲基化率为（4．8±4．5）％；前列腺癌组织中为（48．6±5．3）％，BPH组织中为（4．3±2．1）％，组间比较差异均有统计学意义（P〈0．05）。10μmo1／L5-氮杂胞苷处理LNCaP和DU145细胞72h后，两种细胞中NDRG1基因发生了去甲基化，mRNA表达水平较处理前增强8～9倍，细胞生长受到抑制（P〈0．05）。结论NDRG1基因启动子区的高甲基化是其在前列腺癌中异常表达的原因之-，5-氮杂胞苷能逆转NDRG1基因的甲基化状态，调控该基因mRNA表达，并能抑制前列腺癌细胞的增殖。

Objective To evaluate the methylation status of prostate cancer NDRG1 gene promoter region, and to explore the influence of methylation inhibitor 5-azacytidine on NDRG1 gene＇s mRNA expression in prostate cancer cells and its effects on cell proliferation. Methods Bisulfite-sequencing PCR （BSP） were used to detect the NDRG1 gene promoter methylation status in prostate cancer and BPH tissue, prostate cancer cell lines （PC3,22RV1, LNCaP and DU145 ） and human normal prostate cell line＇s RWPE- 1. After 10 μmol/L 5-azacytidine were used on LNCaP and DU145 cells for 72 h,5-azaeytidine＇s influence on cell proliferation was analyzed with MTT, two prostate cancer cell lines NDRG1 mRNA expressions were detected with RT-PCR. Results The methylation rates of NDRG1 gene in prostate cancer cell lines PC-3, 22RV1, LNCaP and DU145 were （24. 8 ± 3.3 ） %, （36. 2 ±2. 5 ） %, （48.6 ± 2. 8 ） % and （69. 5± 1.7 ） %, respectively. Methylation rate of Human normal prostate cell lines RWPE-1 was （ 4. 8± 4. 5 ） % ; prostate carcinoma was （48.6 ± 5.3）%, BPH tissue was （4. 3 ±2. 1 ）%. The differences between groups were statistically significant. After 10 μmol/L 5-azacytidine added on LNCaP and DU145 cells for 72 h, NDRG1 gene demethylation occurred in both cells, its mRNA expression enhanced 8 -9 times compared with previous and its cell growth was inhibited （P 〈 0.05 ）. Conclusions NDRG1 gene promoter region＇ s hypermethylation is one of the reasons of its aberrant expression in prostate cancer. 5-azacytidine can reverse NDRG1 gene promoter methylation status,regulate the expression of the gene and can inhibit prostate cancer cell proliferation.